Background: Despite the large number of papers published on the
efficiency of different exogenous gonadotropins, no confirmed protocol
exists. Therefore, the aim of the present study was to compare the
efficacy of 4 exogenous gonadotropins in IVF/ICSI cycles.

Methods: This study, performed from January 2014 to May 2014,
recruited 160 women referred to Ghadir Mother and Child Hospital and
Dena Hospital, Shiraz, Iran. The patients underwent standard
downregulation and were randomly divided into 4 groups of A, B, C, and D
and were administered hMG, hFSH, rFSH, and combined sequential
hFSH/rFSH, respectively. Then, the duration of stimulation, number of
oocytes and embryos as well as their quality, implantation rate,
biochemical and clinical pregnancy rate, and live birth rate in each
group were evaluated.

Results: Group D patients required significantly fewer ampoules of
FSH than did the women in groups A, B, and C (P=0.004). The duration of
stimulation was significantly longer in group C than in groups A and D
(P=0.030). The serum estradiol level was significantly higher in group D
than in groups B and C (P=0.005). A significantly higher number of
large-sized follicles was observed in group D than in group B (P=0.036).

Conclusion: Our data revealed no statistically significant
differences in the mean oocyte number, embryo quality, clinical
pregnancy rate, or live birth rate between the hMG, hFSH, rFSH, and
sequential hFSH/rFSH protocols. However, several differences in the
duration of stimulation, serum estradiol levels, and number of
large-sized follicles were detected between the groups.

Today, assisted reproductive technology (ART) has become a
well-established and highly efficient therapy for infertility. In ART,
it is well understood that the most important factors for maximizing the
success rate of in vitro fertilization (IVF) are retrieving greater
numbers of high-quality oocytes using controlled ovarian
hyperstimulation (COH) and establishing a receptive endometrium. (1-4)
Therefore, COH plays a principal role in achieving a high ART success
rate.

Nowadays, the use of long protocols using the
gonadotropin-releasing hormone (GnRH) analog plus gonadotropins for COH
has gained widespread popularity. (5-7) Various gonadotropin
preparations are commercially available and used for COH such as human
menopausal gonadotropin (hMG), human-derived follicle-stimulating
hormone (hFSH), and recombinant FSH (rFSH). hMG contains FSH and
luteinizing hormone activity, while rFSH comprises only FSH, and in
comparison to hFSH, rFSH includes a high proportion of fewer acidic
isoforms with high purity and high in vitro bioactivity. (8) There are
many controversies surrounding which kind of exogenous gonadotropin is
more suitable and leads to greater IVF success.

Some studies have demonstrated that a better outcome in terms of
oocyte and embryo quality, subsequent pregnancy rates, and live birth
rate is obtained when hMG is used for ovarian stimulation, as compared
with rFSH. (9-11) However, other studies have shown that rFSH is as
effective as urinary FSH or hMG in terms of the number of oocytes and
embryos obtained and the total gonadotropin dose needed. (7,9,12)

Studies that compared hFSH with rFSH noted increased ovarian
recruitment of follicles in the rFSH group. (13,14) Daya (12) showed
that rFSH was better than hFSH in terms of the pregnancy rate, while van
Wely et al. illustrated a borderline significant difference of 5% higher
clinical pregnancy rate in women stimulated with hFSH compared with
rFSH. (7) Selman et al. (8) demonstrated that the combination of
hFSH/rFSH for ovarian stimulation had a positive effect on follicular
development, oocyte quality, embryo development, and clinical outcome in
patients with repeated IVF failures.

Therefore, despite the large number of papers published on COH
protocols comparing the efficiency of different exogenous gonadotropins,
no confirmed protocol exists, and it is not quite clear which is
superior to the others. Thus, the objective of the current study was to
compare the efficacy of 4 different ovarian stimulation protocols,
comprising hFSH, rFSH, hMG, and sequential use of hFSH and rFSH, on
oocyte and embryo quality and IVF treatment outcome in patients
undergoing IVF or intracytoplasmic sperm injection (ICSI).

Patients and Methods

Patients

This double-blinded, randomized, clinical trial study was
registered in the Iranian Registry of Clinical Trials (code:
IRCT201408116541N7) and approved by the Institutional Review Board and
the Ethics Committee of Shiraz University of Medical Sciences, Shiraz,
Iran (code: CT-P-92-7249). The study was performed from January 2014 to
May 2014. Written informed consent was obtained from each participant. A
flow chart of the study design is depicted in figure 1. The CONSORT flow
diagram is depicted in figure 2. The sample size used in this study was
determined based on the criteria established by Kutner et al. (15) using
the following formula: type I error ([alpha]) =0.05, power of analysis
(1 - [beta] = 0.95), Effect Size [[micro].sub.1] = [[DELTA]/[sigma]] =
1.0 and number of groups=4

The study group consisted of 160 women referred to 2
hospital-affiliated IVF centers for infertility treatment in Shiraz,
Iran (Ghadir Mother and Child Hospital and Dena Hospital). Patients with
unexplained or male factor infertility were included in the study if
they met the following criteria: 1) age between 20 and 38 years; 2) body
mass index (body weight divided by the square of body height) between 19
and 29 kg/[m.sup.2]; 3) history of regular menstrual cycles, ranging
from 25-35 days; 4) no relevant systemic disease, severe endometriosis,
or uterine or ovarian abnormalities; 5) no more than 3 previous IVF
cycles; and 6) no previous IVF cycle with a poor response or the ovarian
hyperstimulation syndrome. Additionally, patients with FSH >10 IU/mL,
with <5 follicles in antral follicle count, and anti-Mullerian
hormone <1 ng/mL were excluded from the study.

Ovarian Stimulation

After the patients were assessed for eligibility according to the
mentioned criteria, a standard downregulation protocol was performed for
all of them via a subcutaneous injection of GnRH agonists (0.5 mg of
buserelin, Suprefact, Serono), on day 21 of their menstrual cycle (1 wk
before the expected menses). Subsequently, on day 2 of the next
menstrual cycle, after confirming desensitization (estradiol serum
concentration <50 pg/mL, the absence of follicles [greater than or
equal to]10 mm in diameter, and endometrial thickness <5 mm), the
patients were randomized by a person independent of the research team
using a computer-generated random-number list. Thereafter, ovarian
stimulation was commenced for the study population as follows: group A:
40 patients who received hMG (Menogon[R], Ferring Pharmaceuticals A/S,
Copenhagen, Denmark); group B: 40 patients who received hFSH
(Fostimon[R], IBSA Institut Biochimique SA, Geneva, Switzerland); group
C: 40 patients who received rFSH (Gonal-F[R], Merck, Serono, Rome,
Italy); and group D: 40 patients who received hFSH (FostimonX[R], IBSA
Institut Biochimique SA, Geneva, Switzerland) for the first 6 days,
followed by rFSH (Gonal-F[R], Merck, Serono, Rome, Italy). In all the 4
groups, the gonadotropin administration was continued up to the day of
human chorionic gonadotropin injection (hCG) (Gonasi[R] HP, IBSA Italia,
Rome, Italy). It should be mentioned that both the subjects of the study
and the investigators performing the study were blind to the type of the
gonadotropin each patient received for ovarian stimulation.

The monitoring of ovarian responses to gonadotropin stimulation
during the treatment cycle began from day 6, using transvaginal
sonography and the measurement of the plasma E2 level every 3 days. Each
change in the gonadotropin dose was performed according to the follicle
size and the plasma E2 level. The treatment was continued until the
observation of at least 2 follicles having reached 17-18 mm in diameter
(leading follicles) and some other follicles 14-16 mm in diameter. When
the leading follicle was 18-20 mm, and there were at least 3 follicles
of 16-17 mm, gonadotropin administration was stopped, and an
intramuscular injection of 10,000 IU of hCG was administered for final
oocyte maturation. Finally, 34-36 hours after hCG injection,
transvaginal ultrasound-guided oocyte retrieval was performed.

IVF, ICSI, and Assessment of Oocyte and Embryo Quality

Oocyte maturity was tested according to the presence or absence of
a germinal vesicle and first polar body and was graded as GV, MI, or MII
according to the criteria established by Veeck et al. (16,17)
Subsequently, IVF or ICSI, based on indications, was performed. ICSI was
used in the cases with male factor infertility. After fertilization,
embryo scoring was carried out on the day of embryo transfer (3 d after
oocyte retrieval). (16,17) The embryos were graded as I, II, or III,
where I indicates the best-quality embryo and III indicates the
lowest-quality embryo. The luteal phase was supported by an
intramuscular injection of 2 vials of progesterone (50 mg, Iran Hormone,
Tehran, Iran) daily, from the day of oocyte retrieval for 3 days and
continued with intravaginal progesterone (400 mg, Cyclogest[R], Actavis
UK Ltd., Barnstaple, UK) twice per day.

Assessment of Pregnancy, Implantation Rate, and Pregnancy Outcome

Two weeks after embryo transfer, the chemical pregnancy test was
carried out by evaluation of serum [beta]-hCG. In addition, clinical
pregnancy was evaluated by observing the pregnancy sac 6 weeks after
embryo transfer, and the implantation rate was determined by the number
of gestational sacs divided by the number of embryos transferred.

End Points and Outcome Measures

The primary end points were oocyte and embryo quality and pregnancy
outcomes. The secondary endpoints were the duration of stimulation,
plasma E2 level on the day of hCG administration, number of used
ampoules or vials of gonadotropin, number of large-sized follicles,
total number of collected oocytes and transferred embryos, and
implantation and miscarriage rates.

Statistical Analysis

Statistical analysis was performed using SPSS, version 16 (IBM,
Armonk, USA). For the analysis of the data, the one-w ay ANOVA test was
used followed by the Tukey test to compare the means. A P value <0.05
was considered statistically significant.

Results

According to table 1, age, body mass index, duration of
infertility, and endometrial thickness at baseline were similar in all
the groups.

The number of ampoules or vials of gonadotropin administered was
lower in group D than in the other groups; this difference was
statistically significant compared to groups A, B, and C.

The duration of stimulation was longer in group C than in the other
3 groups, and the difference in group C in comparison to groups A and D
was statistically significant.

Endometrial thickness and the estradiol level on the day of hCG
administration were higher in group D than in the other groups. Apropos
the estradiol level, this difference was significant in group D in
comparison to groups A and B.

As is shown in table 2, the number of large-sized follicles was
high in group D and then in group C, compared to groups A and B. This
difference between groups B and D was statistically significant.

The number of retrieved oocytes was higher in groups C and D than
in groups A and B, but the difference did not constitute statistical
significance. The number of degenerated oocytes was higher in group D
than in groups A, B, and C; the difference, however, was not
statistically significant. No statistically significant differences were
observed in the number of GV and MI oocytes between the studied groups,
but the number of mature oocytes (MII) was higher in group C and then in
group D than in groups A and B; nevertheless, the difference was not
statistically significant. The lowest number of MII oocytes was observed
in group B.

According to table 3, the number of transferred embryos was not
different between the groups. The highest proportion of grade-I embryos
and the lowest proportion of grade-II and grade-III embryos were in
group D, followed by groups C, B, and A.

As is shown in table 4, the chemical and clinical pregnancy rate,
implantation rate, and live birth rate were high in group D, followed by
group C, in comparison to the other groups; nonetheless, the difference
was not statistically significant. In addition, the abortion rate was
highest in group D.

Discussion

Among the different protocols for COH, the use of the GnRH analog
plus gonadotropins (long protocol or standard protocol) is popular,
owing to its more favorable results. The literature abounds with studies
comparing exogenous gonadotropins for COH, but the issue still remains
controversial.

Exogenous ovarian stimulation increases oocyte yield but may
compromise the developmental competence of the oocytes in stimulated
cycles. (18) In this study, we evaluated the efficacy of 4 different
ovarian stimulation protocols using different gonadotropins in women
undergoing IVF or ICSI programs.

According to our results, the number of ampoules used was
significantly lower in the sequential protocol than that in the other 3
protocols and the duration of stimulation in the rFSH-alone protocol was
significantly longer than that in the hFSH and hMG protocols. Gerli et
al. (19) demonstrated that stimulation with the sequential protocol,
compared with the rFSH protocol, necessitated a low gonadotropin dose
and short duration of stimulation for the stimulation of ovaries. Other
studies have shown no significant differences between the use of rFSH
and hFSH or the sequential protocol in the duration of stimulation and
the dose of gonadotropin used. (6,13,14,20,21) These contradictory
results may have originated from diversity not only among the products
of pharmaceutical companies but also among patients' race and
physiological status.

The effect of serum estradiol level on the day of hCG on ART
outcome is controversial. It is said that although the estradiol level
increases endometrial proliferation, uterine perfusion, oocyte
development and maturation, number of embryos transferred, implantation,
delivery, and pregnancy rate, the supraphysiological level of estradiol
may not only cause endometrial damage and disrupt the implantation but
also exert negative effects on IVF-ICSI outcome. Nevertheless, this
hypothesis has yet to be confirmed. (22,23)

COH leads to the development of groups of follicles of differing
sizes. Gonadotropin stimulation changes in the steroid profile result in
modifying the microenvironment of the developing follicle and its
oocyte. Precise evaluation of follicle size is highly important, and it
has been shown that larger follicles at the time of retrieval have
consistently mature oocytes with a higher rate of fertilization. (24)

We observed that the level of estradiol was significantly higher in
the sequential protocol than in the hFSH and hMG protocols, resulting in
more large-sized follicles, retrieved oocytes, and MII oocytes in this
protocol. Nonetheless, it did not lead to a clear increase in the
endometrial thickness of these groups compared to the other 2 groups.

The sequential use of hFSH/rFSH is the same as the natural
physiologic cycle, where more acidic isoforms of FSH are produced in the
follicular phase, when the estradiol level is low, and fewer acidic
isoforms are produced in the late follicular and periovulatory phase,
when estradiol is high. (21) The significantly high number of
large-sized follicles in the sequential protocol in comparison to the
rFSH protocol may be related to the combined used of acidic (hFSH) and
less acidic isoforms (rFSH) of FSH, which mimics the physiology of the
normal menstrual cycle and is an important mechanism for the regulation
of the final stages of follicle and oocyte maturation. (25)

Furthermore, in the rFSH protocol, the level of estradiol was
nonsignificantly higher than that with the hFSH and hMG protocols.
Gholami et al. (13) showed a significantly high level of estradiol in
the rFSH protocol compared with the hFSH protocol. Other studies have
shown no differences in the estradiol level and endometrial thickness
between the sequential, rFSH, hFSH, and hMG protocols. (8,19,21,26,27)

Although not significant, the number of retrieved oocytes and MII
oocytes was high in the sequential and rFSH protocols compared with the
hFSH and hMG protocols. However, the number of degenerated oocytes was
nonsignificantly high in the sequential protocol compared to the other 3
groups. In other studies, no significant differences in the number of
retrieved oocytes have been seen between the different protocols.
(8,19,27,28) We observed no significant differences in the number of
retrieved oocytes and mature oocytes between the hFSH and rFSH patients,
chiming in with other studies. (6,21) Gerli et al. (19) observed that
the number of MII oocytes was significantly higher in the patients who
received the sequential protocol than in the patients who received rFSH
alone.

Therefore, using a sequential protocol, our patients reached higher
estradiol levels and sufficient numbers of suitable follicles with fewer
ampoules and lower durations of stimulation. Furthermore, in the rFSH
protocol, despite the need for more ampoules and a longer duration of
stimulation than in the other groups, more retrieved oocytes and higher
numbers of MI and MII oocytes were produced than with the hMG and hFSH
protocols, although these differences were not significant.

We observed that the use of the sequential and rFSH protocols, by
comparison with the hMG or hFSH protocol, nonsignificantly led to more
good-quality (grade I) embryos. In addition, the number of low-quality
embryos was lowest in the sequential protocol, followed by the rFSH,
hFSH, and hMG protocols. Selman et al. (8) and Gerli et al. (19) showed
that the number of good-quality embryos was significantly high in the
sequential protocol in comparison to the hFSH and rFSH protocols. In
other studies, the number of good-quality embryos is similar in the
rFSH, hMG, and hFSH protocols. (13,24,27)

Although the total number of transferred embryos was not different
between the groups, the implantation and pregnancy and live birth rates
were higher in the sequential protocol. This may be related to the
higher number of good-quality embryos produced in the patients who
received the sequential protocol. Selman et al. (8,21) and Gerli et al.
(19) showed that the implantation rate and pregnancy and delivery rates
were significantly high using the sequential protocol in comparison to
the hFSH and rFSH protocols. In the rFSH protocol, these parameters were
slightly higher than in the hFSH and hMG protocols. Gholami et al. (13)
and Selman et al. (26) reported that the implantation rate and pregnancy
rate were similar between the rFSH and hFSH protocols. In contrast, Daya
(12) demonstrated that rFSH was better than hFSH in terms of the
pregnancy rate, while van Wely et al. (7) showed a significantly high
clinical pregnancy rate with the hFSH protocol compared with rFSH.
Ludwig et al. (27) and Turhan et al. (5) showed that the pregnancy and
live birth rates were similar between the rFSH and hMG protocols. These
differences may be due to the heterogeneity of patients in the analysis,
their age, type of GnRH analog suppressions, gonadotropin doses, etc.
Nonetheless, the results of the study by Selman et al. (8) and our
results showed that the sequential protocol was better than the other
protocols in terms of clinical pregnancy and the live birth rate. Our
results regarding the superiority of rFSH over hFSH differed from their
results. In our study, the pregnancy rate was higher in the sequential
and rFSH protocols, although the abortion rate was higher in these
protocols as well; however, the overall outcome (the live birth rate)
stood higher in these 2 groups (not significantly). Accordingly, we
concluded that the rFSH and sequential hFSH/rFSH protocols yielded more
mature oocytes, but the sequential protocol was more valuable in terms
of embryo quality, as was seen in implantation, pregnancies, and live
birth rate. Still, there was no clear difference between the hMG and
hFSH protocols.

The sequential use of hFSH/rFSH is the same as the natural
physiologic cycle, where more acidic isoforms of FSH are produced in the
follicular phase, when the estradiol level is low, and fewer acidic
isoforms are produced in the late follicular and periovulatory phase,
when estradiol is high. This may be an important mechanism for the
regulation of the final stages of follicle and oocyte maturation.
(21,25) Therefore, the difference and distribution of exogenously
applied gonadotropins should be determined and used for ovarian
stimulation.

Obviously, these differences in the effect of FSH isoforms on
follicular development patterns strongly suggest that oocyte development
is also likely to be influenced, that normal follicle development and
ultimately normal oocyte function depend on an appropriate balance of
sequential differentiation, and that this balance is strongly influenced
by FSH isoform distribution. (28)

Conclusion

In conclusion, the sequential protocol was able to improve the
success rate of ART and could, as such, be deemed a valuable protocol in
IVF programs. Further large randomized trials are needed to yield a
precise estimation of any difference between the above-mentioned
protocols.

Acknowledgement

This research was partially extracted from a thesis written by
"Solmaz Rezaee", MD, supported by Shiraz University of Medical
Sciences with the grant number of 7249.